ABSTRACT Melanoma is the sixth most common cancer in the United States, causing almost 8000 deaths annually. While melanoma is treatable when caught in the early stages, once the disease has progressed and becomes metastatic it is almost universally fatal within 5 years, so new therapeutic options are clearly needed. Circulating tumor cells (CTCs) are cancer cells shed from a primary or metastatic tumor and eventually seed new cancerous sites. In melanoma, CTCs have been found to be responsible for much of the aggressive nature of metastatic disease, including resistance to molecularly-targeted therapies. Because the virulence of individual CTCs varies, we hypothesize that unique gene expression signatures in individual CTCs is the source of drug resistance. Specifically, we hypothesize that the BRAF inhibitor vemurafenib imposes a selective pressure on melanoma CTCs which results in resistance. Unraveling the underlying mechanisms of vemurafenib resistance will require comprehensive analysis of individual CTCs during the evolution of resistance. We recently advanced technology to the point where we can robustly isolate rare melanoma CTCs and transcriptionally profile individual CTCs by single cell RNA-seq. With our innovative approaches, we can identify distinct genetic programs in melanoma CTC each with unique potential to acquire resistance to molecularly-targeted therapies. We propose to precisely annotate specific gene expression signatures in individual CTCs to the evolution of vemurafenib resistance. Melanomas are both genetically heterogeneous and clinically accessible and therefore represent an optimal cancer type for the development of analytic methods to assess the effects heterogeneous transcriptional outputs on the evolution of drug resistance. Our investigative team is ideally suited to carry out the technology development and data generation required. Historically, melanoma CTCs have been difficult to isolate and therefore a challenge to study. However, we have overcome this technical hurdle and recently determined optimal conditions for collecting melanoma CTCs from the bloodstream. For this proposal, we seek to leverage our experience, resources and capabilities to develop and implement transcriptional profiling by RNA-seq from individual CTCs isolated from melanoma patients before and after vemurafenib treatment. We will thus define transcriptional outputs in response to vemurafenib treatment, experimentally validate their roles in establishing vemurafenib resistance and thereby identify key genes as potential targets for novel vemurafenib-resistance therapy.